Investigation of N, N, N-Trimethyl-L-alanyl-L-proline Betaine (TMAP) as a Biomarker of Kidney Function

Abstract

Glomerular filtration rate (GFR) is the most widely used tool for the measurement of kidney function, but endogenous biomarkers such as cystatin C and creatinine have limitations. A previous metabolomic study revealed N,N,N-trimethyl-L-alanyl-L-proline betaine (TMAP) to be reflective of kidney function. In this study, we developed a quantitative LCMS assay for the measurement of TMAP and evaluated TMAP as a biomarker of GFR. An assay to measure TMAP was developed using liquid chromatography-mass spectrometry. After validation of the method, we applied it to plasma samples from three distinct kidney disease patient cohorts: nondialysis chronic kidney disease (CKD) patients, patients receiving peritoneal and hemodialysis, and living kidney donors. We investigated whether TMAP was conserved in other mammalian and nonmammalian species, by analyzing plasma samples from Wistar rats with diet-induced CKD and searching for putative matches to the m/z for TMAP and its known fragments in the raw sample data repository "Metabolomics Workbench". The assay can measure plasma TMAP at a lower limit of quantitation (100 ng/mL) with an interday precision and accuracy of 12.8 and 12.1%, respectively. In all three patient cohorts, TMAP concentrations are significantly higher in patients with CKD than in controls with a normal GFR. Further, TMAP concentrations are also elevated in rats with CKD and TMAP is present in the sap produced from Acer saccharum trees. TMAP concentration is inversely related to GFR suggesting that it is a marker of kidney function. TMAP is present in nonmammalian species suggesting that it is part of a biologically conserved process.

Figure 1

Concentration of TMAP (ng/mL), creatinine (μmol/L), and cystatin C (mg/L) in human control, CKD, and ESKD patients from LHSC. (A and B) ** p < 0.01, *** p < 0.001 for multiple comparisons one-way Kruskal–Wallis ANOVA with Dunn’s post-hoc test. (C) ****p < 0.0001 for multiple comparisons one-way ANOVA with Tukey’s post-hoc test. Error bars represent mean ± SD.

Figure 2

Linear regression for inversely transformed TMAP (A, B), creatinine (C, D), and cystatin C (E, F) measurements against eGFR for the LHSC and mGFR for the RDH cohort, respectively, has a coefficient of correlation r² = 0.9464 (A) and r² = 0.5308 (B), r² = 0.9526 (C) and r² = 0.6087 (D), r² = 0.9692 (E) and r² = 0.7506 (F), respectively. Dashed lines represent the 95% confidence interval.

Figure 3

TMAP (A, B), creatinine (C, D), and cystatin C (E and F) concentrations in plasma from recruitment visit to 1 year later in control patients with normal GFR (A, C, E) and living kidney donors (D, E, F). A paired t-test shows a significant difference between timepoints for creatinine (n = 10), TMAP (n = 9), and cystatin C (n = 7) concentrations in living kidney donors ***p < 0.001, ****p < 0.0001.

Figure 4

Concentration of TMAP (ng/mL) in male Wistar rats with adenine-induced CKD, in comparison to controls. Control (n = 8), 0.5% adenine CKD (n = 8), and 0.7% adenine CKD (n = 8). Error bars represent mean ± SD. ***p < 0.001, ****p < 0.0001 for multiple comparisons one-way ANOVA with Tukey’s post-hoc test.

Figure 5

Extracted ion chromatogram of 142.0862 (5 ppm) from MS/MS of 229.1 at 50 NCE for a 5 μg/mL TMAP standard (A) and a sap extract from A. saccharum (C). The product ion spectra of the TMAP standard (B) and a sap extract from A. saccharum (D) show the presence of additional product ions at the same relative ion ratios supporting that TMAP is present within this plant-based sample.